Efficiency Measurements for Chromatography Columns - Using the method of moments provides a better characterization of column effluent curves than the frequently used Gaussian approximation. -

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Efficiency Measurements for Chromatography Columns
Using the method of moments provides a better characterization of column effluent curves than the frequently used Gaussian approximation.


BioPharm International
Volume 18, Issue 8

These differences provide a very powerful tool for determining the quality of the flow distribution. Visualize a perfectly packed column with perfect headers, operated under normal conditions until a tracer pulse is halfway to the normal outlet, and then the flow is reversed — the effluent will now appear at the inlet. The pulse will have the same form as that leaving the full column under normal operation — small-scale contributions to band broadening will be the same as for normal operation. However, as per Equation (21), even if there is large-scale maldistribution of flow, the reversed-flow effluent will be that of a perfectly packed column with perfect headers.


Figure 5. Comparison of Forward and Reverse-flow of an Acetone Pulse The conventional forward flow and reverse flow effluent curves have a plate count of 548 and 788. respectively.
A comparison of a pulse injected under normal conditions and under flow reversal at one-half of the retention time is shown in Figure 5. Flow reversal at one-half of the retention time mimics the effluent distribution of a perfectly packed column with perfect headers. The conventional, or forward-flow, effluent curve exhibits significant tailing for the system and column, which is not seen under reverse-flow conditions. This chromatographic column is only operating at 70% of its potential efficiency as determined by a comparison on the number of plates between the forward-flow and the reverse-flow case.

A further extension of this reverse-flow technique has been developed that allows for the decoupling of the effects of non-uniform packing from poor distributor flow, with the latter solely dictated by the column design.12 This extension provides a non-destructive test to characterize flow distribution.

FRONTAL ANALYSIS

In the majority of columns, performance is evaluated only once after the column is packed. Columns are often used multiple times, and it is essential to maintain packed- bed quality and efficiency throughout the column lifetime. Performing tracer analysis to measure the number of plates before every run can be time consuming and impractical for pilot- or commercial-scale columns. Frontal analysis, where a step change is applied to the packed bed rather than a pulse, is frequently used to estimate column performance without the need for extra lines or extra processing time.


Figure 6. Comparison of Column Performance as Determined from the Response to a Salt Pulse and a Salt Front. Column 10 29 cm PhenylSepharose FF(High Sub). Salt Pulse Conditions: Mobile Phase: 400 mM Citrate, Solute Pulse, 50 mL of 150mM Citrate. Salt Front Conditions: Mobile Phase: 1: 400 mM Citrate, Mobile Phase II, 150 mM Citrate. Chromatograms rescaled to overlay.
It is easy to apply the established plate theory for linear chromatography to the response from a pulse. For linear systems, the response to a pulse input is equal to the derivative of the response to a step input.13 Thus numerical differentiation of the monitored output will provide the same information as a response to a non-interacting tracer pulse. This is shown in Figure 6, where the response to a pulse and the differentiated response to a step are overlaid. Both chromatograms show similar responses, particularly in the peak front. The differentiated step response shows a slight deviation at the tail, which would result in a slightly higher HETP.


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